Detection of PML/RARA Fusion Protein in APL Using Proximity Ligation Assay as an Alternative to FISH Testing
Juraj Bodo, Jeffrey J Lin, Jarek P Maciejewski, Andrew E Schade, Eric D Hsi. Institute of Pathology and Laboratory Medicine, Cleveland Clinic, Cleveland, OH; Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH; Diagnostics Research/Development, Eli Lilly and Company, Indianapolis, IN
Background: The presence of t(15,17)(q22;q12) resulting in PML/RARA fusion in acute promyeolocytic leukemia (APL) cases determines eligibility for immediate specific therapy. Currently, FISH and qPCR may be used to distinguish APL from other types of AML. We have developed a brightfield, slide-based method for the rapid in situ detection of the PML/RARA fusion protein using proximity ligation assay (PLA) and evaluated the correlation between PML/RARA fusion protein expression in APL and PML/RARA rearrangement.
Design: A total of 10 AML and APL cytospin samples with known PML/RARA FISH status were stained using PLA (OLINK Bioscience, Uppsala, Sweden) with PML and RARA specific antibodies. The principle of PLA is the recognition of target by two antibodies that can bring into proximity oligonucleotides that are conjugated to secondary antibodies. The oligos participate in ligation, creating a template for generating a large, tethered DNA molecule by amplification and detected with labeled hybridization probes, followed by brightfield detection.
Results: PLA was optimized with the NB-4 cell line (APL cell line with PML/RARA fusion protein) and SU-DHL-6 cell line (DLBCL cell line lacking PML/RARA fusion protein, but expressing normal PML and RARA). All 5 PML/RARA FISH positive APL cases were positive for PML/RARA expression by PLA. The majority of tumors cells were positive in PML/RARA rearranged cases (Figure 1). Importantly, there was no false positive staining among the 5 AML cases lacking t(15;17).
Conclusions: Early detection of PML/RARA is critical for directing appropriate therapy in APL. Our PLA specifically identified PML/RARA+ APL cases and can be performed and analyzed without specialized equipment. This is proof of principle that PLA can be used to detect clinically relevant fusion proteins with brightfield microscopy within a few hours.
Figure 1. Detection of PML/RARA fusion protein expression by PLA. NB-4 (1) and SU-DHL-6 (3) cells served as positive and negative controls, respectively. Examples of PML/RARA+ APL (2) and PML/RARA- AML (4) samples are shown.
Monday, March 4, 2013 1:00 PM
Poster Session II # 269, Monday Afternoon